Synthetic oxygen transport made from cross-linked modified human or porcine haemoglobin with improved properties, method for a preparation thereof from purified material and use thereof

ABSTRACT

According to the claims, the present invention comprises the preparation of chemically modified, cross-linked hemoglobins with improved functional properties, the cross-linked hemoglobins prepared according to this method and the use of these hemoglobins as artificial oxygen carriers. The synthesis method is characterized by technical simplicity as well as by high yields.  
     Deoxygenated hemoglobin of high purity is conjugated covalently under the protection of an antioxidant with an effector of oxygen binding, especially with pyridoxal-5-phosphate, after which the hemoglobin is polymerized with glutardialdehyde as a bifunctional cross-linking agent. At the same time, there is a large increase in the volume of the reaction mixture and a decrease in the concentration of the hemoglobin during the addition of the cross-linking agent. Subsequently, after further dilution with water, a polyethylene oxide derivative is chemically linked to the cross-linked hemoglobins. Polymers are obtained, which are compatible with blood plasma and have optimized cooperativity and half saturation pressure values and can find use as artificial oxygen carriers and, in particular, are divided into a lower molecular weight fraction as blood substitute and a higher molecular weight fraction as blood additive, for example, for the treatment of oxygen-deficiency conditions.

[0001] In accordance with the claims, the present invention comprisesthe production of chemically modified, cross-linked hemoglobins withimproved functional properties, the cross-linked hemoglobins produced bythis method and their use as artificial oxygen carriers. The productionmethod is characterized by its technical simplicity as well as by highyields.

[0002] Deoxygenated hemoglobin of high purity is conjugated covalentlyunder the protection of an antioxidant with an effector for oxygenbonding, especially with pyridoxal-5-phosphate. After that, and thehemoglobin is polymerized with glutardialdehyde with a very largeincrease in the volume of the reaction mixture and, accordingly, a verygreat dilution of the reactants during the addition of the cross-linkingagent. Subsequently, after dilution with water, a polyethylene oxidederivative is linked chemically to the cross-linked hemoglobins.Polymers with optimized oxygen-binding characteristics are obtained,which are compatible with blood plasma and, especially when divided intoa low molecular weight and a high molecular weight fraction, can be usedas artificial oxygen carriers as a blood substitute or blood additive,for example, for the treatment of oxygen deficiency conditions.

[0003] For various clinical indications in medicine, it is desirable tohave available an artificial support system for the transport of oxygen.In the event of an acute loss of blood, it is not only appropriate toreplace the liquid volume isotonically and isocontically, but also torestore a further essential function of the blood, namely the transportof oxygen. With the decreasing preparedness to donate blood, adequatesupplies of blood are available less and less in the event of an acutecatastrophe or in the event of a war, particularly for covering anunforeseeable demand. The instantaneous availability of suitablesupplies of blood furthermore is associated with appreciable logisticproblems. Moreover, blood can usually be stored for only about 35 daysand must therefore be constantly replenished. This creates appreciablecosts. On the other hand, artificial solutions can be kept for asignificantly longer time, since they may optionally be frozen.Depending on the storage time, stored blood acidifies intracellularly.As a result, its acute oxygen binding characteristics are not by anymeans optimum and must be regenerated once again in the organism. On theother hand, an artificial oxygen carrier functions optimally from thefirst instant. The increasing lack of preparedness to donate blood iscontrasted, on the other hand, by the increase in demand of an agingpopulation. At the same time, because of the aging of the population,the number of potential blood donors has decreased. Likewise, because ofunpredictable risks of infection (immune weakness, hepatitis), thepopulation of the slums is unavailable as blood donors. An artificial,oxygen-transporting blood replacement would also be universal andindependent of the blood group. Moreover, it is possible that a volumedeficiency shock can be counteracted more rapidly with such a bloodreplacement than with stored blood, since the erythrocytes in the storedblood have stiffened and therefore have a lower capillary permeability.In any case, animal experiments have shown that a volume deficiencyshock can be combated more effectively with an oxygen-transporting bloodreplacement than with simple plasma expanders (Pabst, R. (1977): “OxygenTransport with Stroma-free Hemoglobin Solutions and Fluorocarbons”, Med.Klin. 72: 1555-1562, Keipert, P. E., Chang, T. M. S (1985):“Pyridoxylated Polyhemogloblin as a Red Cell Substitute forResuscitation of Lethal Hemorrhagic Shock in Conscious Rats: Biomater.,Med. Dev., Artif. Organs 13: 1-15). There are other applications for anartificial oxygen carrier, such as complicated surgical interventions,which necessarily are associated with high blood losses and can becarried out less and less, because of a lack of appropriate storedblood. On the other hand, larger and more invasive surgicalinterventions, including also transplants, are constantly beingdeveloped. In a particular case, the possibility of carrying out such anintervention depends decisively on the availability of a sufficientnumber of suitable stored bloods. Moreover, organs, which are to betransplanted, can be preserved far better, if they are perfused with(artificial) oxygen carriers. For instance, a liver transplant requiresup to 100 transfusion units of 450 mL each. In cases of polytraumaticinjuries, such as those caused by an automobile accident, similarlylarge amounts are required.

[0004] However, there is a need for an artificial oxygen-transporting,blood additive not only in the case of an acute loss of blood, but alsoin the case of chronic blood-circulation disorders (especially cerebral,coronary, renal and peripheral—such as a sudden loss of hearing—or ananemic crisis, such as in the case of chronic osteomyelitis or aftertumor chemotherapy). The use of the additive is also an option in orderto prevent a threatening abortion in the case of a fetal oxygendeficiency due to a placenta insufficiency or to preventoxygen-deficiency injuries during birth. A requirement of this type iseven significantly greater than that in the aforementioned case of anacute blood loss. Such chronic circulation disorders are the cause ofdeath of about 750,000 people annually in Germany. In addition, thereare the cases of illness that arise from this cause. On the other hand,only about half that number of people die from cancer every year. It iswell known that attempts are made to treat chronic oxygen deficienciesin tissues by supplying hyperbaric oxygen. Aside from the fact that thistreatment is effective only as long as the oxygen overpressure existsand the procedure is not harmless, the danger of oxidative damage totissue due to reactions of free radicals of oxygen, which can bedetected by corresponding reaction products, exists here. An artificialoxygen carrier acts as long as it is present and offers so-calledlow-pressure oxygen to the tissue, so that the aforementioned damagedoes not occur.

[0005] The use of the oxygen-transporting blood additives as a temporarysupport for the endogenous oxygen transport system offers a furtherpossibility and alternative for combating a chronic tissue deficiency ofoxygen, the treatment of which was previously attempted withcirculation-promoting means, such as vessel dilators.

[0006] The fact that it is a functional oxygen treatment is very much tothe benefit of the concept. It is not the substrate (the oxygen) that isimproved; instead, the function of the carrier system, which brings theoxygen to the tissue is improved. This makes the treatmentmultiplicative in its effect and, with that, very effective and givesit, at the same time, a catalytic character. In addition, there areclear indications that an oxygen carrier, which is dissolved in plasma,is far more effective than one, which is “packed” in erythrocytes.

[0007] Moreover, such an artificial blood replacement can be producedfree of known pathogens; infection problems, such as hepatitis andacquired immune deficiency (AIDS) are avoided in this manner.

[0008] An additional potential group of recipients for artificialoxygen-transporting solutions are patients, for whom an allergicreaction, for example, to HLA antigens, may be expected. Until now, theremoval of leukocytes from stored blood by filtration through cotton hasbeen attempted. On the other hand, artificial blood replacementsolutions would be completely free of leukocytes. Recently, it has beenobserved in pigs that, after an improvement in the oxygen supply(decrease in the oxygen affinity of the hemoglobin), the cardiac outputof the heart is decreased while the heart rate remains unaffected(Vilereal M. C., et al. (1987): “Engineered Red Blood Cells withModified Oxygen-Transport Properties: A New Oxygen Carrier”, Biomater.,Med. Dev., Artif. Organs 15: 397). In a different paper (Bosman, R. J.,et al. (1992): “Free Polymerized Hemoglobin Versus Hydroxyethyl Starchin Resuscitation of Hypovolemic Dogs”, Anesth. Analg. 75: 811-817”, itwas shown that the administration of oxygen-transporting solutions aftera volume deficiency shock in dogs prevented the increase in the cardiacoutput and accordingly is easy on the heart. By improving the oxygensupply, the possibility arises of achieving a functional cardiacprotection, which is helpful, for example, in the case of an infarction.This would be a completely new aspect of the use of oxygen-transportingsolutions.

[0009] A further use of such artificial oxygen carriers would be toincrease in the radiation sensitivity of tumors, especially since thereare increasing indications that molecular oxygen carriers, dissolved inthe plasma, deliver the oxygen to the tissue far more effectively thandoes whole blood. Such artificial carriers bring about a synergism withthe native (intraerythrocytic) carrier. In other words, the molecularlydispersed artificial carrier in the blood plasma not only per se is bestat giving off oxygen from the capillary, but also intensifies the oxygendelivery of the existing native system by way of the mechanism offacilitated diffusion. This means that only a very low concentration ofthe artificial carrier in the plasma is required for this purpose.Nevertheless, this functional therapy (see above) remains extremelyeffective.

[0010] Everything, which has been stated, makes clear the need for anartificial oxygen transporter. It is indispensable for the use of anartificial oxygen carrier that its starting material is available insufficient amounts. Deteriorated stored blood accordingly does not solvethe problem. It is therefore necessary to use animal hemoglobins,preferably from the most important slaughtered animals, namely cattleand/or pigs.

[0011] Roughly two types of applications arise from the presentation ofthe need for artificial oxygen carriers, on the one hand, in the case ofa heavy loss of blood and, on the other, in the case of a chronic oxygendeficiency. In the first case, an iso-oncotic, oxygen transportingvolume substitute (artificial oxygen carrier of the first generation) isrequired as compensation and on the other hand, in the second case, anoxygen-transporting blood additive (artificial oxygen carrier of asecond and new generation) is required. As already mentioned, the lattercase is far more frequent. Moreover, an appropriate blood additive, incombination with so-called plasma expander, also permits an acute lossof blood to be treated with the great advantage that the physician hasthe possibility of coordinating the administration of oxygen carriers aswell as the liquid volume with the requirements with the individualpatient.

[0012] The organism is also able to change both (the amount of oxygencarrier and the blood volume) independently of one another, namely theerythrocyte formation by way of the erythropoetin and the plasma volumeby way of its own regulation system. The two parameters are uncoupledowing to the fact that the carrier has a much smaller colloidal osmoticpressure than does the plasma.

[0013] Previously, three basically different strategies for developingan artificial oxygen carrier for blood were employed by others (state ofthe art: Rudolph, A. S. et al. (Publisher) Red Blood Cell Substitutes:Basic Principles and Clinical Applications, Marcel Dekker, New York, etal., 1998; Tsuchida E. (Publisher): Blood Substitutes: Present andFuture Perspectives, Elsevier Science, Amsterdam, 1998; Chang, T. M. S.(Author & Publisher) Blood Substitutes: Principles, Methods, Productsand Clinical Trials, Volume 1 and Volume 2, Karger Landes, Basel et al.1997 and 1998).

[0014] The use of emulsions with fluorinated hydrocarbons—recently,other halogens, such as bromine, have also been used—in which oxygen isparticularly soluble (Hirlinger, W. K., et al. (1982): Effects of aPartial Blood Exchange with Fluosol DA 20% on the intact organism of thepig”, Anästhesist 31 660-666). However, since the fluorinatedhydrocarbons are lipophilic, it is to be expected that interactions anddisorders in the lipid layers of the cell membrane will occur. The laterare integrating, functional components of the cell. Moreover, thefluorinated hydrocarbons must be disposed with emulsifiers, such asphospholipids, which can interfere additionally with the membranes ofthe cells (so-called artificial oxygen carriers, based on fluorinatedhydrocarbons of the first generation).

[0015] A further strategy is represented by the microencapsulation ofhighly concentrated solutions of natural and also of chemically modifiedhemoglobins in phospholipid vesicles with addition of suitable effectorsof hemoglobin binding (“artificial erythrocytes or hemosomes”) (Ogata Y.(1994): “Characteristics of Neo Red Cells, Their Function and Safety:In-Vivo Studies”, Artificial Cells, Blood Substitutes, andImmobilization Biotechnologies 22: 875-881). The first animalexperiments in this field have been successful (Hunt, C. A. et al.(1985): “Synthesis and Evaluation of a Protypal Artificial Red Cell”,Science 230: 1165-1168). The vesicles had a diameter of less than 0.05μm and their volume therefore was more than two powers of ten smallerthan that of natural red blood cells.

[0016] The third strategy consists of the production of infusiblehemoglobin solutions. The artificial oxygen carrier then occursextracellularly in the blood. The first two solutions of the problem canbe regarded as relating to the production of an artificial blood, sothat the difficulty of a colloidal, osmotic interference cannot occur.On the other hand, for this solution of the problem, there was a plasmaexpander at the very start, the macromolecules of which can alsotransport oxygen (a so-called, artificial oxygen carrier), on the basisof hemoglobin of the first generation.

[0017] Native hemoglobin cannot be used for this purpose, for example,because it is eliminated too rapidly by way of the kidneys. A chemicalmodification therefore is indispensable. For example, within the scopeof this strategy, hemoglobin has been bound by its amino groupscovalently to dextrans or has itself been polymerized to a molecularweight of 700,000 g/mole. The former procedure was pursued, for example,by Firma Fresenius (Fresenium E. (1976), Blood and PlasmaSubstitute—Comprising a Colloidal Solution of Hydroxyethyl StarchCoupled to Hemoglobin-Free Stroma”, Patent DE-P 2616-086) and the laterby Firma Biotest (Bonhard K., et al. (1983): “Method for ObtainingHepatitis-Safe, Sterile, Pyrogen-Free and Stroma-Free HemoglobinSolutions”, Patent DE-O 31 30 770) and Firma Alza (Bonsen P. (1976):“Water-soluble Polymerized Hemoglobin”, Patent DE-O 26 07 706). Afurther strategy with regard to extracellular solutions is thestabilization of hemoglobin by cross-linking it to an intratetramer orby appending side groups (oligo ethylene glycol) without significantlyincreasing the molecular weight of the tetramer (stabilized hemoglobins(Matsushita M. et al. (1987): “In Vivo Evaluation of PyridoxylatedHemoglobin-Polyoxythene Conjugate”, Biomat, Artif. Cells. Artif. Org.15: 377). As mentioned above, extracellular blood replacement solutionshave given promising results with respect to treatment of shock inanimal experiments.

[0018] The use of hemoglobin as an artificial oxygen carrier has anadvantage over the use of fluorinated hydrocarbons, because theadvantageous properties of the natural binding of oxygen can beutilized. These include the optimally adapted oxygen affinity, thehomotropic cooperativity, that is, the S-shape of the oxygen bindingcurve, as well as the (alkaline) drilling effect which forms the basisof a natural, self-regulatory mechanism for the selective delivery ofoxygen to deficiently supplied tissue.

[0019] It is clearly evident from the relevant literature that anintra-tetrameric, covalent bonding of hemoglobin units (Keipert P. E.,et al. (1989): “Metabolism, Distribution, and Excretion of HbXL: ANondissociation Interdimerically Crosslinked Hemoglobin with ExceptionalOxygen Offloading Capability”,—in: Change, T. M. S., Geyer R. P. (Eds.):Blood Substitutes, Marcel Dekker, New York 1989) and/or a polymerizationof the hemoglobin leads to a large increase in the residence time in theblood. (Chang T. M. S. (1987): “Modified Hemoglobin as Red Cell BloodSubstitutes”, Biomater., Med. Devices Artif. Organs 14: 323-328;Friedman H. J., et al. (1984): “In Vivo Evaluation ofPyridoxylated-Polymerized Hemoglobin Solution”, Surg. Gynecol., Obstet.:159 429-435). This is an essential perquisite for the clinicalusefulness of such solutions.

[0020] However, in the case of extracellular, molecularly disposedartificial oxygen carriers, a very large area of need, the chronicoxygen deficiency, has been ignored by aiming for iso-oncotic solutions.As already mentioned, the significantly more frequently occurringconsequences of chronic circulation disorders can, however, be improvedonly by oxygen-transporting solutions, the colloidal osmotic pressure ofwhich can be disregarded with respect to the normal (35 mbar), that is,only with the help of an oxygen-transporting blood additive, a“molecular erythrocytes concentrate”, as it were. These are artificialoxygen carriers based on hemoglobin of a second generation.

[0021] The following problems arose during the different attempts todevelop an artificial oxygen transporter:

[0022] Increase in the hemoglobin affinity for oxygen: due to thechemical modification of the hemoglobin molecule, the half saturationpressure (P50) is decreased. As a result, the delivery of oxygen to thetissue is made more difficult. This occurs in a pronounced manner duringthe binding of hemoglobin to dextran. In order to avoid the increase inoxygen affinity, suitable effectors (such as pyridoxal phosphate) havebeen linked to the prosthetic group of the hemoglobin.

[0023] Frequently, the so-called n50 value (HILL index) as an expressionof the decreased homotropic cooperativity (weakened S shape of theoxygen-hemoglobin binding curve), is decreased at the same time; thisalso makes it more difficult to supply the tissue with oxygen. This Sshape of the oxygen-hemoglobin binding curve at the same timefacilitates the absorption of oxygen in the lung and its delivery to thecells. On the other hand, fluorinated hydrocarbons have a linear “oxygenbinding curve” and therefore do not have this functional advantage.

[0024] The artificial oxygen carrier frequently has too short aresidence time in the organism, the dissolved hemoglobins beingeliminated by way of the kidneys. In the case of extracellularhemoglobin solutions as artificial oxygen carriers, the attempt has beenmade to prevent the elimination by intermolecular cross-linking;nevertheless, the residence time of the extracellular hemoglobins remainshorter than desired. On the other hand, hemosomes are removed from theplasma by the reticuloendothelial system of the organism. For example,the half life of the artificial erythrocytes (see above) was 5.8 hours.

[0025] Excessive colloidal osmotic pressure: This can result in a lossof volume (volume deficiency shock). This effect occurs when themolecular weight of the artificial oxygen carrier is comparable to thatof the plasma proteins. As a result, the dosage of the artificial oxygencarrier cannot be chosen freely and, instead, the oncotic relationshipsmust be taken into consideration.

[0026] The oncotic milieu of the plasma continues to be determinedprimarily by the so-called second virial coefficient (A₂ value). Thischaracterizes the interaction of the (always macromolecular) oxygencarrier with the solvent (water). The synthesis should be conducted sothat this value is close to zero.

[0027] Excessive viscosity of the carrier solution: Usually, this isassociated with an A₂ value, which is too large, and it occurspreferably when a carrier consists of chain molecules. According to theEinstein viscosity law, an excessive viscosity does not occur if thepolymeric carrier molecules are spherical and compact.

[0028] In vitro stability of the carrier molecules: This refers, on theone hand, to the decomposition of the molecules and, on the other, tothe oxidative binding of methemoglobin, which is no longer capable ofbinding oxygen, and finally to the viscosity due to the slowly changinginteractions between the carrier and the albumin of the plasma.

[0029] Excessive reaction of the reticuloendothelial system (RES): Themain influencing factor is the molecular size of the artificial carrier.The critical limit for this is about 0.3 μm. Larger particles activatethe RES.

[0030] Kidney and liver damage: a kidney shock occurs especially if thestroma-containing hemoglobin solution is used. Ever since the solutionswere subjected to ultrafiltration, kidney damage has no longer beingobserved. Liver damage was indicated with the help of the plasmatransaminase mirror; it is presumably based on cellular membraneinteractions: the liver has an open flow path (fenestrated capillaries).

[0031] Hemostasis must also be checked: disorders in the sense ofpreventing and inhibiting hemostasis are conceivable; special attentionmust be paid to thrombocyte aggregation.

[0032] Antigenic effects: In this connection, it was recently shown inhomolog experiments in rats that native hemoglobin has no antigenicactivity and that the polymerization with glutardialehyde has notincreased the antigenicity (Hertzman, C. M., et al. (1986): “SerumAntibody Titers in Rats Receiving Repeated Small Subcutaneous Injectionsof Hemoglobin or Polyhemoglobin: A Preliminary Report”, Int. J. Artif.Organs 9: 179-182). In the same paper it is shown that native andpolymerized human hemoglobin has little antigenic activity in rats andthat the effect is intensified at most slightly by polymerization.

[0033] Toxic effects can be differentiated as pyrogenic,vasoconstrictive—for example, on coronary vessels—and endotoxic. Thevasoconstrictive effect is based presumably on entrapping nitrogenmonoxide radicals, which are, and is well known, endogenous,vasodilatoric control substances; however, the vasoconstrictive effectcan also be explained by the high effectiveness of the artificialcarrier, in that the smooth musculature is supplied “too well” withoxygen.

[0034] In vivo stability: An endogenous, enzymatic degradation, forexample, by proteases, must be considered.

[0035] Overloading the organism with lipoids (emulsifiers): thiscomplication occurs only when microencapsulated hemoglobin solutions offluorinated hydrocarbons are used; a complement activation by way of thealternative path as well as antibody formation was noted here.

[0036] Compatibility of the artificial carriers with fresh, human bloodplasma, there may be precipitation, depending on the pH.

[0037] Presumably, because of the problems listed here, an artificialoxygen carrier is as yet not available for routine clinicalapplications. It is evident from the various problems named that ausable artificial oxygen carrier must still satisfy a larger range ofrequirements.

[0038] In nature, oxygen is always transported in microscopic giantaggregates. Two basically different “problem solutions” have beendeveloped here. In the case of higher animals (and also in man), themolecular oxygen carrier is packed in cells (in the erythrocytes).Secondly, oxygen-binding giant molecules develop, which are dissolvedextracellularly in a hemolymph and not intracellularly. This variationis encountered predominantly in lower elements to different degrees.Annelides, for example, have high molecular weight hemoglobins,averaging about three million g/mole, as oxygen carrier. Furthermore,there are so-called hemerythins, for which the iron, as oxygen binder,is released molecularly directly with the protein. Finally, there arethe hemocyanins with a molecular weight of about eight million g/mole,for which copper is the oxygen-binding heavy metal ion (see alsoBarnikol, W. K. R., et al. (1996): “Hyperpolymeric Hemoglobins andArtificial Oxygen Carriers. An Innovative Attempt at MedicalDevelopment”, Therapiewoche 46: 811-815). The historical transition fromextracellular dissolved oxygen carriers to the erythrocytes has causedan increase in the oxygen content of the blood fluid by a factor ofthree. For example, 1 mL of the “blood” of the earthworm can bind atmost 3.6 μmoles of oxygen. On the other hand, up to 9.0 μmoles of oxygenare bound in the same volume of human blood.

[0039] The earthworm has giant molecules (erythrocruorin) in its bloodas oxygen carrier with a molecular weight of about 3,400,000 g/moles andapproximately 200 binding sites for oxygen. Moreover, the molecule isvery compact and its quaternary structure is highly ordered. Themolecule is so large, that it can be made visible directly with the helpof the electron microscope. Its concentration in the hemolymph is atleast 6 g/dL. Measuring the oxygen-binding curve under simulated in vivoconditions gives a half saturation pressure (P50) of 9.1 torr (Barnikol,W. K. R. and O. Burkard (1987): “Highly Polymerized Human Hemoglobin asan Oxygen-Carrying Blood Substitute”, Advances in Experimental Medicineand Biology, vol. 215: 129-134; Barnikol, W. K. R. (1986) “The Influenceof Glutardialdehyde on the Oxygen Cooperativity of Human Hemoglobin”,Pflügers Archiv 406: R 61). This system thus supplies the cells of theearthworm adequately with oxygen. Due to the extremely high molecularweight, the hemoglobin of the earthworm has practically no colloidalosmotic effect anymore (only about 0.4 mbar). With that, as in the bloodof mammals—the oncotic pressure of the erythrocytes is only 10⁻⁷torr—the two functions of colloid osmolarity and oxygen binding areuncoupled and both can be varied freely as control variables of theorganism.

[0040] If the principle of the earthworm system is to be transferred toartificial oxygen carriers, which are based on human or animalhemoglobin, the hemoglobin molecule must be changed so that, asextracellular giant molecule with a negligible oncotic pressure, it cansupport the normal oxygen transport of the erythrocyte at leastintermittently. Such artificial oxygen carriers then are highlypolymerized hemoglobins, which must take into account all the problemsmentioned above. Earthworm “hemoglobin”, which admittedly fulfills theoncotic pressure requirement, will not be usable for man for thispurpose, because it presumably has too high an antigenicity; moreover,the half-saturation pressure of 9 torr is too low (Barnikol, W. K. R andO. Burkard (1987): “Highly Polymerized Human Hemoglobin as anOxygen-Carrying Blood Substitute”, Advances in Experimental Medicine andBiology, vol. 215: 129-134; Barnikol, W. K. R. (1986): “The Influence ofGlutardialdyde on the Oxygen Cooperativity of Human Hemoglobin”,Pflügers Archiv 406: R 61). Moreover, it can presumably not be obtainedin the amounts required.

[0041] Until now, in the development of artificial oxygen carriers,attempts were made to adjust the half saturation pressure of thecarriers precisely to the normal value of about 20 torr in man. However,animal experiments showed that a molecularly disperse artificial oxygencarrier with a half saturation pressure of about 15 torr oxygenatesorgans best (Conover et al. (1999), Art. Cells, Blood Subst. Immobil.Biotech. 27: 93-107). On the other hand, however, animal experimentsalso show that a sufficiently large oxygen capacity of the blood is atleast equally important for an adequate oxygen supply (Moss, G. S., etal (1984): “Hemoglobin Solution—From Tetramer Polymers”, In: The RedCell: Sixth Aven. Arbor Conference, Alan R. Riss, N.Y., 1984: 191-210).In turn, this depends on the possible concentration of the oxygencarrier in the plasma. Furthermore, it was shown here that it is notnecessary to adhere to a half saturation pressure of 26 torr. Rather, itis essential that a certain critical value must be exceeded.

[0042] It is a further requirement of the development of an artificialoxygen carrier that a manufacturing process be as simple as possibleand, with that, economic, especially since it is necessary to work understerile conditions from the start. The yield of the product should behigh and material for use as blood additive and material for use asoxygen-transporting blood-volume substitutes should be formedsimultaneously during the cross-linking reaction.

[0043] Furthermore, during the production of the artificial polymersfrom hemoglobin by linking the hemoglobin molecules over their aminogroups by means of suitable, bifunctional cross-linking agents,particular attention must be paid to the fact that molecular networks,which are insoluble and therefore decrease the yield, are not formed.For this reason, the formation of the so-called percolation distributionof the molecular weight is to be prevented.

[0044] It is therefore an object of the present invention to be able toproduce hypo-oncotic, artificial oxygen carriers from cross-linkedhemoglobins, which have optimized, good functional properties,especially the characteristics of oxygen binding, and are suitable foruse as pharmaceutical products in man, in a technically simple processin a large yield.

[0045] Pursuant to the invention, this objective is accomplished asdescribed below. Surprisingly, it was possible eliminate the fundamentalproblem of the formation of a percolation distribution of themultimerization degrees and molecular weights by the cross-linking ofthe polyfunctional hemoglobins with the bifunctional cross-linkingagent, glutardialdehyde, by greatly increasing the volume of thereaction mixture (2- to 10-fold in all) during the cross-linking, and,moreover, adding initially the cross-linker as a dilute solution andsubsequently additionally diluting with water. By these means,cross-linked hemoglobins with a high degree of cross-linking are formedin a large yield, without developing a percolation distribution of themolecular weight and forming insoluble, molecular networks. Rather, thecrude product of the cross-linking is distinguished by a certain,desirable (approximate) upper limit of the molecular weight range. Aso-called quadrilateral distribution of the molecular weight is obtainedin the gel chromatogram (see FIGS. 1, 2 and 3).

[0046] The inventive preparation thus comprises the following steps:

[0047] In a one-vessel reaction, hemoglobin

[0048] i) initially is deoxygenated, especially by passing nitrogen overit,

[0049] ii) subsequently reacted covalently with a chemically reactiveeffector of the oxygen binding,

[0050] iii) after which the solution is treated with a chemicallyunreactive effector and then

[0051] iv) the hemogloblin is cross-linked stably and covalently withglutardialdehyde with a very great dilution (2- to 5-fold of the volumeof the reaction mixture, with simultaneous addition of the cross-linker(in solution)) and subsequently the reaction volume is increased furtherwith water, the total dilution of the solution being 2- to 10-fold,especially 2- to 7-fold and particularly 5- to 6-fold and then

[0052] v) a polyethylene oxide is linked covalently.

[0053] The product obtained can be worked up by known methods.

[0054] Preferably, the starting hemoglobin originates from pigs or fromman, pigs and especially domestic pigs being particularly preferred.

[0055] Particularly and pursuant to the invention, the glutardialdehydeis added in step iii) in a very highly diluted solution in atime-controlled manner. Preferably, there is a subsequent furtherdilution with water and an increase in the reaction volume, so that theabove-mentioned total dilution is achieved.

[0056] Moreover, in step iv), glutardialdehyde is preferably added in amolar amount of 6 to 10 and especially of 7 to 9 moles/mole, based onthe monomeric hemoglobin, dissolved in 1-4, preferably 1-2, particularly1.5-2 and especially 1.7-1.9 L of water per liter of original reactionsolution.

[0057] This addition is timed to take place between about 3 and 15,especially between 3 and 10 and particularly between 4 and 6 minutes.Subsequently, the solution is reacted for 1 to 6 hours.

[0058] It is furthermore preferred that, before the reaction of stepii), 2-8, especially 3-6 and particularly 3-4 moles of sodium ascorbateper mole of uncrosslinked hemoglobin be added to the solution containingthe hemoglobin. This reaction takes place over a period of 0.5 to 6hours and especially of 70 to 120 minutes.

[0059] Furthermore, in step ii), preferably pyridoxal-5′-phosphate islinked as effector in a molar ratio, based on the monomeric hemoglobin,of 0.5 to 3, preferably of 1 to 2.5 moles/mole covalently over a periodof 0.5 to 20 and especially of 1 to 7 hours.

[0060] Pursuant to the invention, and furthermore preferably andadvantageously, reductive sodium borohydride is added in step ii) aswell as in step iv) after the covalent linking of pyridoxal-5′-phosphateand of glutardialdehyde.

[0061] This is added particularly in step ii) in an amount of 1 to 9,preferably of 1 to 5 and particularly of 1 to 2.5 moles/mole, forexample, over a period of 30 to 90 minutes, and in step iv) in an amountof 5 to 20 and especially of 6 to 12 moles/mole, in each case based onthe monomeric hemoglobin, over a period of 15 to 100 minutes.

[0062] It is particularly preferred if, as unreactive effector in stepiii), 2,3-bisphosphoglycerate is added in an amount of 0.5 to 6 andespecially of 1 to 4 moles/mole, based on the monomeric hemoglobin, andif about 5 to 50 minutes, especially 10 to 20 minutes and particularly15 minutes thereafter glutardialdehyde is added.

[0063] As polyethylene oxide, preferably a polyethylene glycol etherwith, for example, a C1-C5 alkyl group, such as methyl, ethyl and butyl,with a molecular weight of 500 to 3000 g/mole, especially amethoxy-polyethylene glycol derivative with a molecular weight of 1500to 2500 g/mole and especially of 2000 g/mole, such as, especially,methoxypolyethylene glycol succinimidyl propionate, in amounts of 2 to12 and especially 3 to 8 moles/mole of hemoglobin. Other derivatizingproducts are methoxy-polyethylene glycol succinimidyl succinamide andmethoxy-polyethylene glycol succinimidyl hydroxyacetate. The linking ofpolyalkylene oxide to uncrosslinked hemoglobin is described in U.S. Pat.Nos. 4,179,637, 5,478,805 and 5,386,014 and the EP-A 0 206 448, EP-A 0067 029 and the DE OS 3,026,398.

[0064] The reaction is carried preferably over 1 to 4 and particularlyover 2 to 3 hours.

[0065] It is furthermore advantageous that all synthesis reactions takeplace, in particular, in solutions freed from oxygen by tonometry withoxygen-free gases. The method used is described by H. Potzschke in“Hyperpolymers of Human Hemoglobin, Development of Preparative Methodsfor their Synthesis, Validation of Analytical Methods and Equipment fortheir Characterization”, Dissertation, Medical Faculty, University ofVienna, 1997.

[0066] The product obtained can be worked up in the usual manner, asdescribed in the following. In particular, it has a molecular weightdistribution of 50,000 to 5,000,000 and optionally up to 10,000,000g/moles or more.

[0067] Preferably, the product obtained can be divided into a fractionof high average molecular weight and a fraction of low average molecularweight, the boundary preferably being at 700,000 g/moles, by apreparative, material-separating method, such as a preparative volumeexclusion chromatography, an ultrafiltration, a fractionalprecipitation, for example, with polyalkylene oxide or salt such asammonium sulfate as precipitant, or a field-flow fractionation method(Curling, J. M (publisher), “Methods of Plasma Protein Fractionation”,Academic Press, London et al. 1980, as well as the patents EP-A 0 854151 and EP-A 95 107 280).

[0068] A pharmaceutical preparation can be produced from the product ofhigh molecular weight and also from the product of low molecular weight,a parenteral blood substitute being obtained from the low molecularfraction of the polymers and a parenteral blood additive from the highmolecular weight fraction of the polymers.

[0069] Before (and after) the individual steps of the reaction, the pHis adjusted preferably with lactic acid or sodium hydroxide solution tovalues between 6 and 10, depending on the reaction step, for example, toa value of 6.5 to 7.5 before step ii), subsequently to 7.5 to 8.5 andsubsequently again to 6.5 to 7.5 before step iii), after that to 7.5 to9, as well as to 7.5 to 10 before step v).

[0070] The concentration of hemoglobin preferably is 200 to 380 g/L andparticularly 240 to 360 g/L; the solution furthermore contains 10 to 150mmoles/L of sodium bicarbonate and 10 to 150 mmoles/L of sodiumchloride.

[0071] During the “one-vessel reaction”, the temperature is 2° to 42°C., especially 3° to 25° C. and particularly 4° to 24° C.

[0072] The artificial oxygen carrier, produced pursuant to theinvention, preferably has an n50 value (cooperativity) of 1.6 to 2.5 anda p50 value (semi-saturation pressure) of 16 to 24 torr.

[0073] The product, which is obtained pursuant to the invention and hasthe characteristics given, can be used to produce an agent for anantivasal or biomedical application as an artificial oxygen carrier, orin the form of a pharmaceutical preparation as a blood substitute or asan additive to blood to increase the oxygen transport capacity (bloodadditive) or to a nutrient solution in human and animal organisms, inindividual organs or in biotechnical applications, especially for thetreatment of a chronic oxygen deficiency in man.

[0074] To prepare the products, which are to be administered, theinventive hemoglobin derivatives are dissolved in suitable media, suchas infusion solutions, for example in aqueous salt or glucose solutions,preferably in concentrations isotonic with the blood plasma.

[0075] The inventive method is based accordingly on individual reactionsteps, which are matched to one another and the significance and effectsof which are explained in the following.

[0076] The starting material is a very pure hemoglobin. This may beobtained by known methods from the fresh blood of slaughtered animalsor, for example, overaged stored blood. Methods for obtaining purifiedhemoglobins are described by Antonini, E. et al. (publisher) in:“Hemoglobin”? (Colowick, S. P. and N. O. Kaplan (publishers): Methods inEnzymology, Vol. 76) Academic Press, New York et al. 1981.

[0077] As mentioned, pursuant to the invention, during the cross-linkingwith glutardialdehyde, which is known (DE 24 99 885, U.S. Pat. Nos.4,857,636, 4,001,200, 4,001,401 and DE 449 885, U.S. Pat. No. 5,439,882,EP A 0 221 618), the hemoglobin is deoxygenated (that is, freed from itsphysiological ligands, oxygen), because only polymers, which areprepared from hemoglobin that is deoxygenated during the cross-linkinghave the oxygen binding properties, which enable them to be used asartificial oxygen carriers for the desired indications. Preferably, asfurther protection against oxidation of the hemoglobin by traces ofremaining oxygen, the latter can be removed by chemical reaction withascorbate ions.

[0078] Especially pyridoxal-5′-phosphate is linked as effector of theoxygen binding in an amount, optimized for the functional properties ofthe end product, covalently to the pig or human hemoglobin. This linkingof pyridoxal-5′-phosphate to hemoglobins basically is known; forexample, patent EP-P 0 528 841 describes a method for the synthesis ofpyridoxylated hemoglobin. The pyridoxylation leads to the desiredhalf-saturation pressure value if the inventive procedure is employed.

[0079] The linking sites (aldimines=Schiffs bases) of the unstablecovalent linkage of pyridoxal-5′-phosphate can be stabilized, as is wellknown (see above), by reduction with sodium borohydride (amines areformed), the aforementioned special conditions being adhered to pursuantto the invention. The capability of hemoglobin molecules for homotropiccooperativity of the oxygen binding sites with one another is generallyclearly lost by cross-linking the hemoglobin with the cross-linkingagent, glutardialdehyde. Pursuant to the invention, and to maintain thiscapability, 2,3-bisphosphoglycerate, a “heterotropic”, chemically notreactive effector of the oxygen binding of the hemoglobin, is addedbefore the cross-linking. Accordingly, this 2,3-bisphosphoglycerate canbe added reversibly to its binding site in the hemoglobin moleculeduring the cross-linking. After the synthesis, 2,3-bisphosphoglycerate,together with unused reactants as well as excess reaction product, isremoved completely (see below).

[0080] Glutardialdehyde is used as bifunctional cross-linking agentunder the inventive conditions given above.

[0081] Two cross-linking sites (aldimines=Schiffs bases) of thecrosslinked molecular glutardialdehyde bonds are stabilized, asdescribed, by reduction with sodium borohydride (amines are formed), theinventive conditions being observed.

[0082] Cross-linked hemoglobins are formed by the cross-linking and havea molecular weight distribution between about 50,000 and 5,000,000g/mole (and larger, for example, 10,000,000 to 15,000,000 g/mole).

[0083] To improve the compatibility with plasma proteins, a polyethyleneoxide (MW) derivative, especially the above-mentioned, monofunctional,activated (methoxy) polyethylene glycol having a molecular weight of1,500 to 2,500 g/mole, is linked to the cross-linked hemoglobin. Thelinking of polyethylene glycol (PEG), the so-called pegylation, to aswell as the cross-linking of hemoglobins is known (see also E. Tsucheda(Publisher); Blood Substitutes; Present and Future Perspectives,Elsevier Science, Amsterdam 1998).

[0084] However, the pegylation, as well as the cross-linking ofhemoglobins together, as well as the use of the chemically inactiveeffector before the cross-linking, which contributes particularly toobtaining cooperativity as well as, in particular, the cross-linkingconditions described, are new. The anyhow weak reaction of the RES andalso the enzymatic degradation by proteases are prevented by thepegylation carried out here.

[0085] The pH is adjusted as described above.

[0086] On the one hand, the development of a percolation distributioncan be prevented by the inventive dilution and by the reaction sequenceand conditions obtained. On the other hand, undesirable changes in theoxygen affinity and cooperativity can be avoided by the cross-linking ofand covalent attachment to hemoglobin by, for example, glutardialdehyde,pyridoxal phosphate and polyethylene oxide and, in particular, a highplasma compatibility can also be achieved.

[0087] All reaction steps together contribute to these specialproperties of the product produced pursuant to the invention.

[0088] The further working up is within the scope of knowledge ofsomeone skilled in the art. Insoluble components can be removed bycentrifugation (for example, for 20 minutes with a relative centrifugalacceleration of 20,000 g). The cross-linked hemoglobins are divided by a“well known” preparative step of the process, especially by a volumeexclusion chromatography or ultrafiltration, a factional precipitationor a field flow fractionation into a higher molecular weight and a lowermolecular weight fraction. If suitable methods are selected, (forexample, the nominal molecular-weight separation boundary of theultrafiltration membrane or the molecular weight separation range of thegel used are particularly important), the unused reactants as well asthe undesirable reaction products are removed at the same time.

[0089] A particularly preferred embodiment of the invention is explainedin greater detail by means of the following preparation example.

[0090] Purified pig or human hemoglobin, with a concentration between200 and 380 g/L and preferably between 240 and 360 g/L, is dissolved inan aqueous electrolyte. The latter contain sodium hydrogen carbonate ata concentration between 10 and 150 mmoles/L and preferably between 40and 60 mmoles/L, and sodium chloride at a concentration of between 10and 150 moles/L and preferably between 50 and 100 mmoles per L. Thetemperature is 2° to 42° C. and preferably between 3° and 25° C. Thehemoglobin solution is stirred and pure nitrogen is passed over it inorder to deoxygenate the hemoglobin. To this solution, 2 to 8 moles andpreferably 3 to 4 moles of sodium ascorbate (as a 1 molar solution inwater) are added per mole of hemoglobin and allowed to react for aperiod between 0.5 and 6 hours and preferably between 70 and 120minutes.

[0091] The pH of the solution is now adjusted to a value between 6.5 and7.5 and preferably between 6.9 and 7.3 with lactic acid or sodiumhydroxide solution having a concentration between 0.1 and 1 andpreferably between 0.4 and 0.6 moles/L. Pyridoxal-5′-phosphate (0.5 to3.0 and preferably from 1.0 to 2.5 moles per mole of hemoglobin) is nowadded and allowed to react between 0.5 and 20 and preferably between 1and 7 hours.

[0092] The pH is now adjusted with sodium hydroxide solution or lacticacid, having a concentration between 0.1 and 1 and preferably between0.4 and 0.6 moles/L to a value between 7.5 and 8.5 and preferablybetween 7.7 and 8.2.

[0093] Now, 1.0 to 9.0 and preferably 1.0 to 2.5 moles of sodiumborohydride (as a 1-molar solution in 0.01 molar sodium hydroxidesolution) are added and allowed to react for between 30 and 90 andpreferably for between 50 and 70 minutes

[0094] The pH of the solution is adjusted with lactic acid or sodiumhydroxide solution, having a concentration between 0.1 and 1 andpreferably between 0.4 and 0.6 moles/L) to a value between 6.5 and 7.5and preferably between 6.9 and 7.5

[0095] Now, 0.5 to 6.0 and preferably 1.0 to 4.0 moles of2,3-bisphosphoglycerate per mole of hemoglobin are added and allowed toreact for between 5 and 50 and preferably for between 10 and 20 minutes.

[0096] Subsequently, the time-controlled addition of the bifunctionalcross-linking agent is made. Between 6 and 10 and preferably between 7and 9 moles of glutardialdehyde per mole of hemoglobin, dissolved in 1to 4 and preferably 1.5 to 2 L of water per 1 of hemoglobin solution,are added within 3 to 10 and preferably within 4 to 6 minutes andallowed to react for between 1 and 6 and preferably for between 2 and 3hours.

[0097] The pH is adjusted with sodium hydroxide or lactic acid, having aconcentration between 0.1 and 1 and preferably between 0.4 and 0.6moles/L, to a value between 7.5 and 9.0 and preferably between 7.6 and8.8.

[0098] In all, 5 to 20 and preferably 6 to 12 moles of sodiumborohydride (as a 1-molar solution in 0.01 molar sodium hydroxidesolution) is added per mole of hemoglobin and allowed to react forbetween 15 and 100 and preferably between 30 and 80 minutes. This isfollowed by the addition of 0 to 4 and preferably between 0.5 and 3 L ofwater per liter of the original hemoglobin solution.

[0099] If necessary, the pH is adjusted with sodium hydroxide solutionor lactic acid, having a concentration between 0.1 and 1 and preferablybetween 0.4 and 0.6 moles/L to a value between 7.5 and 10 and preferablybetween 8 and 9. Preferably, between 3 and 8 moles of an activatedpolyethylene oxide derivative, preferably methoxy-succinimidylpropionate polyethylene glycol, with a molecular weight between 500 and3000, preferably 1000 and 2500 and particularly 2000 g/mole are nowadded per mole of monomeric hemoglobin.

[0100] Subsequently, while continuing to stir the hemoglobin solution,the nitrogen atmosphere is replaced for 1 to 3 hours by pure oxygen andthe hemoglobin thus is oxygenated swiftly. The working up is carried outas described above.

[0101] The advantages of the inventive method can be summarized asfollow.

[0102] Due to the inventive reaction sequence, especially the increasein the reaction volume in step iv) of the method, a product is obtained,which can be used completely for the preparation of artificial oxygencarriers. Moreover, about half of the product can be used as a bloodadditive (the fraction with the crosslinked hemoglobins of higherdegrees of polymerization, fraction I) and as blood volume substitute(fraction II), with the low molecular weight fractions. The separationcan be accomplished easily with known methods, some possible methodsbeing, for example, listed in the patents EP-A 95 107 280 and EP-A 97100 790. The polymers of fraction I, preferably up to a molecular weightof more than 700,000 g/mole, are so adequately uniform on a molecularbasis, that they have an adequately low colloidal osmotic pressure inthe desirably therapeutic plasma concentration. A small viralcoefficient as well as a low viscosity are achieved by this molecularuniformity. The comparability of the proteins of the blood plasma, thesufficiently large immune compatibility and intravasal residence time,as well as adequately low vasoconstrictive side effects, that is, a lowextravasation, of the polymers of fraction I is achieved by a covalentlinkage of the polyalkylene oxides. Moreover, the requirement ofsimplicity and economic efficiency is taken into account in a decisivemanner by this new method in that the whole preparation takes place in asingle vessel (a so-called one-vessel preparation) and in high yields ofmore than 70%, the yield of polymers with a molecular weight of morethan 700,000 g/mole exceeding 15%.

[0103] The method makes it possible to prepare modified and cross-linkedhemoglobins in a few process steps. The process parameters selected leadto a defined distribution of modified hemoglobin polymers, which, asartificial oxygen carriers, suitably take into account also thephysiological circumstances in the blood serum.

[0104] Moreover, the cooperativity of the uncrossed-link hemoglobin inthe cross-linked product is largely obtained and the half saturationpressure can be adjusted suitably.

[0105] In parenteral application, the artificial oxygen carriers,produced pursuant to the invention from cross-linked hemoglobin, arecompatible with the plasma and can be employed clinically as described.

[0106] The invention is described in greater detail by means of thefollowing examples, FIGS. 1 to 3 showing the following:

[0107]FIG. 1 shows a mass average distribution of the molecular sizesand molecular weights (M) of the pig hemoglobin polymers of Example 1,shown as a volume exclusion chromatogram, obtained with a SephacrylS-400 HR gel, Pharmacia, Freiburg, Germany. E425 nm is the extinction inthe chromatography eluate. The abscissa values of 700,000 and 5,000,000g/mole are shown.

[0108]FIG. 2 shows a chromatogram for Example 2, the explanations beinggiven in FIG. 1.

[0109]FIG. 3 shows a chromatogram for Example 3, the explanations beinggiven in FIG. 1.

[0110] Furthermore, the following analytical methods were employed.

[0111] 1. The hemoglobin contents were determined photometrically withthe modified cyanhemoglobin method of Drabkin (“Hemoglobin Color TestMRP 3”, Boehrimger Mannheim, Germany).

[0112] 2. pH values were measured potentiometrically (glass electrode)with a blood-gas analyzer (“ABL 5”, Radiometer, Willich, Germany)

[0113] 3. The molecular weight distribution of the cross-linkedhemoglobins was determined by volume-exclusion chromatography (PötzschkeH. et al. (1996): “Cross-linked Globular Proteins—A new class ofsemi-synthetic polymeric molecules: characterization of their structureand solution in solution by means of volume-exclusion chromatography,viscosimetry, osmometry and light scattering using hyperpolymerichemoglobins and mycoglobins as example”, Macromolecular Chemistry andPhysics 197, 1419-1437, as well as Pötzschke H. et al. (1996): “A novelmethod for determining molecular weights of widely distributed polymerswith the help of gel photography and viscosimetry using hemoglobinhyperpolymers as examples”, Macromolecular Chemistry and Physics 197,3229-3250) on Sephacryl S-400 HR gel (Pharmacia Biotech, Freiburg,Germany).

[0114] 4. The characteristics of oxygen binding by hemoglobins weredetermined by means of our own methods and equipment (as described inBarnikol W. K. R. et al. (1978): “An improved modification of the Nieseland Thews micromethod for measuring O₂—Hb-binding curves in whole bloodand concentrated Hb solutions”, Respiration 36, 86-95).

[0115] 5. The Plasma compatibility of cross-linked hemoglobins wasinvestigated by means of a standardized in vitro precipitation test(Domack, U. (1997), “Development and in vivo evaluation of an artificialoxygen carrier on the basis of bovine hemoglobin”, dissertationChemistry Department of the Johannes Gutenberg University, Mainz 1997):Hemoglobin solutions (with hemoglobin contents of about 30 g/L in anaqueous electrolyte (StLg) containing 125 mM of sodium chloride, 4.5 mMof potassium chloride and 3 mM of sodium nitride) were mixed with equalamounts of freshly obtained, sterile filtered, human plasma.Subsequently, up to 20 μL of a 0.5 molar lactic acid was added to ineach case 500 μL of the mixture and mixed in, so that for eachhemoglobin derivative, which was to be investigated, the pH ranged from7.4 to 6.8. After an incubation period of 30 minutes at room temperatureand centrifugation of the samples, the hemoglobin content was determinedas a measure of the hemoglobin, which had not been precipitated, and theassociated pH in the supernatant was determined. In addition, asubjective, visual check for colorless precipitate of plasma proteinswas conducted.

EXAMPLE 1 Preparation of an Inventive Cross-Linked and MolecularlyModified Pig Hemoglobin at 4° C. by the General Method Of Preparation

[0116] Pig hemoglobin of high purity, dissolved in a concentration of330 g/L in an aqueous electrolyte containing 50 mM of sodium bicarbonateand 100 mM of sodium chloride, was deoxygenated at 4° C. by stirring thesolution under pure nitrogen, which was constantly being renewed.Subsequently, 4 moles of sodium ascorbate (as a I-molar solution inwater) was added per mole of (monomeric) hemoglobin and allowed to reactfor 6 hours. The pH of the solution was adjusted to a value of 7.1 with0.5-molar lactic acid. Subsequently, 1.1 mole of pyridoxal-5′-phosphatewere added per mole of hemoglobin and allowed to react for 16 hours. ThepH of the solution was now adjusted to a value of 7.8 with 0.5 molarsodium hydroxide solution and 1.1 moles of sodium borohydride (as a1-molar solution in 0.01-molar sodium hydroxide solution) were added andallowed to react for 1 hour. The pH was now adjusted to a value of 7.3with 0.5-molar lactic acid. Initially, 1.1 moles of2,3-bisphosphoglycerate per mole of hemoglobin and, after a reactionperiod of 15 minutes, 8 moles of glutardialdehyde per mole ofhemoglobin, dissolved in 1.8 L of pure water, was added per liter ofhemoglobin solution for cross-linking the hemoglobin within 5 minutesand allowed to react for 2.5 hours. After the pH was adjusted to a valueof 7.8 with a 0.5 molar sodium hydroxide solution, 15 moles of sodiumborohydride (as a 1-molar solution in 0.01 molar sodium hydroxidesolution) were added per mole of hemoglobin for 1 hour. Subsequently, 2L of water were added per liter of original hemoglobin solution. The pHthen was 9.3. Immediately thereafter, 4 moles of methoxysuccinimidylpropionate polyethylene glycol having a molecular weight of 2,000 g/molewere added over a period of 2 hours. The nitrogen atmosphere over thesolution was replaced by pure oxygen.

[0117] After 1 hour, the insoluble constituents were removed bycentrifuging (20.000 g for 15 minutes). Subsequently, the electrolytewas changed by a volume exclusion chromatography (Sephadex G-25 gel,Pharmacia, Germany) to an aqueous electrolyte solution containing 125 mMof sodium chloride, 4.5 mM of potassium chloride and 20 mM of sodiumbicarbonate.

[0118] The yield was 77%; the yield for a molecular weight greater than700,000 g/mole is 28%.

[0119]FIG. 1 shows a representation of the distribution of the molarmasses of the hemoglobin polymers obtained in the form of a volumeexclusion chromatogram.

[0120] Measurements of the characteristic of the oxygen binding underphysiological conditions (a temperature of 37° C., a carbon dioxidepartial pressure of 40 torr and a pH of 7.4) reveal that the product hada p50 value of 22 torr and an n50 value of 1.95.

[0121] In the “precipitation test”, the cross-linked pig hemoglobin, inthe pH range of 7.4 to 6.8, showed no interactions whatsoever with humanplasma, especially no detectable precipitates of the hemoglobin or ofthe plasma proteins.

EXAMPLE 2 Preparation of an Inventive Cross-Linked and MolecularlyModified Pig Hemoglobin at Room Temperature by the General Method ofPreparation

[0122] Pig hemoglobin of high purity, dissolved in a concentration of330 g/L in an aqueous electrolyte containing 50 mM of sodium bicarbonateand 100 mM of sodium chloride, was deoxygenated at 22° C. by stirringthe solution under pure nitrogen, which was constantly being renewed.Subsequently, 4 moles of sodium ascorbate (as a I-molar solution inwater) was added per mole of (monomeric) hemoglobin and allowed to reactfor 90 minutes, the pH of the solution was adjusted to a value of 7.1with 0.5-molar lactic acid. Subsequently, 1.1 moles ofpyridoxal-5′-phosphate were added per mole of hemoglobin and allowed toreact for 2 hours. The pH of the solution was now adjusted to a value of7.8 with 0.5 molar sodium hydroxide solution and 1.5 moles of sodiumborohydride (as a 1-molar solution in 0.01-molar sodium hydroxidesolution) were added and allowed to react for 1 hour. The pH was nowadjusted to a value of 7.3 with 0.5-molar lactic acid. Initially, 1.5moles of 2,3-bisphosphoglycerate per mole of hemoglobin and, after areaction period of 15 minutes, 9 moles of glutardialdehyde per mole ofhemoglobin, dissolved in 1.8 L of pure water, was added per liter ofhemoglobin solution within 5 minutes, for cross-linking the hemoglobinand allowed to react for 1 hour. After the pH was adjusted to a value of7.8 with a 0.5 molar sodium hydroxide solution, 10 moles of sodiumborohydride (as a 1-molar solution in 0.01 molar sodium hydroxidesolution) were added per mole of hemoglobin for 30 minutes. The pH was8.7. Immediately thereafter, 8 moles of methoxysuccinimidyl propionatepolyethylene glycol having a molecular weight of 1000 g/mole, were addedover a period of 1 hour. The nitrogen atmosphere over the solution wasreplaced with pure oxygen.

[0123] After 1 hour, the insoluble constituents were removed bycentrifuging (20.000 g for 10 minutes). Subsequently, the electrolytewas changed by volume exclusion chromatography (Sephadex G-25 gel,Pharmacia, Germany) to an aqueous electrolyte solution containing 125 mMof sodium chloride, 4.5 mM of potassium chloride and 20 mM of sodiumbicarbonate.

[0124] The yield was 79%; the yield for a molecular weight greater than700,000 μmole was 28%.

[0125]FIG. 2 shows a representation of the distribution of the molarmasses of the hemoglobin polymers obtained in the form of a volumeexclusion chromatogram.

[0126] Measurements of the characteristic of the oxygen binding underphysiological conditions (a temperature of 37° C., a carbon dioxidepartial pressure of 40 torr and a pH of 7.4) reveal that the product hada p50 value of 22 torr and an n50 value of 1.6.

[0127] In the “precipitation test”, the cross-linked pig hemoglobin, inthe physiologically and pathophysiologically interesting pH range of 7.4to 6.8, showed no interactions whatsoever with human plasma, especiallyno detectible precipitates of the hemoglobin or the plasma proteins.

EXAMPLE 3 Preparation of an Inventive Cross-Linked and MolecularlyModified Human Hemoglobin at 4° C. by the General Method of Preparation

[0128] Human hemoglobin of high purity, dissolved in a concentration of330 g/L in an aqueous electrolyte containing 50 mM of sodium bicarbonateand 100 mM of sodium chloride, was deoxygenated at 4° C. by stirring thesolution under pure nitrogen, which was constantly being renewed.Subsequently, 4 moles of sodium ascorbate (as a I-molar solution inwater) was added per mole of (monomeric) hemoglobin and allowed to reactfor 3 hours. The pH of the solution was adjusted to a value of 7.1 with0.5-molar lactic acid. Subsequently, 1.1 mole of pyridoxal-5′-phosphatewere added per mole of hemoglobin and allowed to react for 16 hours. ThepH of the solution was now adjusted to a value of 7.8 with 0.5 molarsodium hydroxide solution and 1.1 moles of sodium borohydride (as a1-molar solution in 0.01-molar sodium hydroxide solution) were added andallowed to react for 1 hour. The pH was now adjusted to a value of 7.3with 0.5-molar lactic acid. Initially, 1.5 moles of2,3-bisphosphoglycerate per mole of hemoglobin and, after a reactionperiod of 15 minutes, 9 moles of glutardialdehyde per mole ofhemoglobin, dissolved in 1.8 L of pure water, were added uniformlywithin 5 minutes per liter of hemoglobin solution for cross-linking thehemoglobin and allowed to react for 2.5 hours. After the pH was adjustedto a value of 8.0 with a 0.5 molar sodium hydroxide solution, 10 molesof sodium borohydride (as a 1-molar solution in 0.01 molar sodiumhydroxide solution) were added per mole of hemoglobin for 1 hour.Subsequently, 2 L of water were added per liter of original hemoglobinsolution. The pH then was 8.6. Immediately thereafter, 4 moles ofmethoxysuccinimidyl propionate polyethylene glycol, having a molecularweight of 2000 g/mole, was added over a period of 2 hours. The nitrogenatmosphere over the solution was replaced with pure oxygen.

[0129] After 1 hour, the insoluble constituents were removed bycentrifuging (20.000 g for 10 minutes). Subsequently, the electrolytewas changed by a volume exclusion chromatography (Sephadex G-25 gel,Pharmacia, Germany) to an aqueous electrolyte solution containing 125 mMof sodium chloride, 4.5 mM of potassium chloride and 20 mM of sodiumbicarbonate.

[0130]FIG. 3 shows a representation of the distribution of the molarmasses of the hemoglobin polymers obtained in the form of a volumeexclusion chromatogram.

[0131] The total yield was 75%; the yield of polymers with a molecularweight greater than 700,000 g/mole was 17%.

[0132] Measurements of the characteristic of the oxygen binding underphysiological conditions (a temperature of 37° C., a carbon dioxidepartial pressure of 40 torr and a pH of 7.4 reveal that the product hada p50 value (as a measure of the average oxygen affinity) of 21 torr andan n50 value (an average apparent cooperativity of the oxygen bindingsites) of 1.74.

[0133] In the “precipitation test”, the cross-linked pig hemoglobin, inthe physiologically and pathophysiologically interesting pH range of 7.4to 6.8, showed no interactions whatsoever with human plasma, especiallyno detectible precipitates of the hemoglobin or of plasma proteins.

1. A method for the technically simple preparation of artificial oxygencarriers from cross-linked hemoglobin with improved functionalproperties in a high-yield, wherein the hemoglobin i) initially isdeoxygenated, especially by passing nitrogen over it, ii) subsequentlyreacted covalently with a chemically reactive effector of the oxygenbinding, iii) after which the solution is treated with a chemicallyunreactive effector and then iv) the hemogloblin is cross-linked stablyand covalently with glutardialdehyde with a very great dilution andsubsequently the solution is diluted with water and v) a polyethyleneoxide is then linked covalently. vi) the product obtained is worked upby known methods.
 2. The method of claim 1, wherein the hemoglobinoriginates from pigs or from man.
 3. The method of claim 2, wherein thestarting material is pig hemoglobin.
 4. The method of one of the claims1 to 3, wherein, in step iv), glutardialdehyde is added in atime-controlled manner to a very highly diluted solution and in such away, that the volume of the reaction mixture and the hemoglobinconcentration during the polymerization reaction are variedsimultaneously in opposite directions and the solution subsequently isdiluted.
 5. The method of one of the claim 1 to 4, wherein the volume ofthe reaction mixture increases by a factor of 2 to 10 during step iv).6. The method of claim 4, wherein, in step iv), glutardialdehyde,dissolved in 1 to 2 L of water per liter of original reaction solution,is added over a period of 3 to 15 minutes in an amount of 6 to 10moles/mole, based on the monomeric hemoglobin, and reacted for a further1-6 hours
 7. The method of one of the claims 1 to 6, wherein, inaccordance with step ii), 2 to 8 moles of sodium ascorbate per mole ofuncrosslinked hemoglobin are added over a period 0.5 to 6 hours to thesolution containing the hemoglobin.
 8. The method of one of the claims 1to 7, wherein, in step ii), pyridoxal-5′-phosphate is linked covalently,in a molar ratio, based on the monomeric hemoglobin, of 0.5 to 3 andpreferably of 1 to 2.5 mole/moles, over a period of 0.5 to 20 hours, aseffector.
 9. The method of claim 8, wherein, in step ii) as well as instep iv), reductive sodium borohydride is added after the covalentlinkage of pyridoxal-5′-phosphate to the hemoglobin as well as after thecovalent linkage of the hemoglobin with the glutardialdehyde.
 10. Themethod of claim 9, wherein the reductive sodium borohydride is added fora period of 30 to 90 minutes or a period of 15 to 100 minutes in stepii) in a relative amount of 1 to 9 moles/mole and, in step iv), in anamount of 5 to 20 moles/mole in each case based on monomeric hemoglobin.11. The method of one of the claims 1 to 10, wherein2,3-bisphosphoglycerate is added in step iii) in a relative amount of0.5-6 mole/mole, based on the monomeric hemoglobin, and step iv) isinitiated 5-50 minutes later.
 12. The method of one of the claims 1 to11, wherein a polyethylene glycol ether, with a molecular weight of 500to 3,000 g/mole is linked in step v).
 13. The method of claim 12,wherein a methoxy polyethylene glycol derivative with a molecular weightof 1,500 to 2,500 is linked as polyethylene glycol ether.
 14. The methodof one of the claims 1 to 13, wherein the synthesis reactions take placein solutions freed from oxygen by tonometry with oxygen-free gases. 15.The method of one of the claims 1 to 13, wherein all steps of the methodcan be carried out consecutively in a single vessel.
 16. The method ofone of the claims 1 to 15, wherein a product is obtained, which has amolecular is distribution from 50,000 to 5,000,000 g/mole.
 17. Themethod of one of the claims 1 to 16, wherein the total yield is inexcess of 70% and the yield of polymers with a molecular weight of morethan 700,000 g/mole is greater than 15%.
 18. The method of one of theclaims 1 to 17, wherein the product obtained is divided by a preparativeseparation method into a fraction with a larger average molecular weightand a fraction with a smaller average molecular weight.
 19. The methodof claim 18, wherein a pharmaceutical preparation is prepared from theproduct with the higher molecular weight and also from the product withthe lower molecular weight.
 20. The method of claim 19, wherein aparenteral blood substitute is prepared from the low molecular weightfraction of the polymers.
 21. The method of claim 19, wherein aparenteral blood additive is prepared from the higher molecular weightfraction of the polymers.
 22. An artificial oxygen carrier, prepared bythe method of one of the claims 1-21.
 23. An artificial oxygen carrierof claim 22, wherein the carrier has an N50 value of 1.6 to 2 to 5 and ap50 value of 16 to 24 torr.
 24. The use of cross-linked hemoglobin ofclaims 22 or 23 or prepared by the method of one of the claims 1 to 22for the preparation of an agent for the intravasal or biomedicalapplication as an artificial oxygen carrier.
 25. The use of claim 24,wherein the agent is used in the form of a pharmaceutical preparation asa replacement for blood (blood substitute) or as an addition to theblood for increasing the oxygen transport capacity (blood additive) orto a nutrient, in the human and animal organism, in individual organs orin biotechnical applications.
 26. The use of claims 23 or 24, for thetreatment of a chronic oxygen deficiency in man.